Symposium summary

Veterinary Parasitology, 10 (1982) 113--118 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

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SYMPOSIUM SUMMARY

In a review, Gasbarre and Urban address cellular immune responses elicited by parasitic infections. They discuss problems associated with, and advancements of techniques for the assessment of T l y m p h o c y t e responses induced by parasite antigens. Accurate assessments of T cell activity are required at both the inductive phases (stimulation of T cells by parasite antigens) and effector phases (responses of sensitized i m m u n o c o m p e t e n t cells). Modified blastogenesis assays utilize T cell growth factor (TCGF) in techniques of cloning and continuous in vitro propagation of antigenreactive T cells. Responses can be quantified by enumeration of immunoc o m p e t e n t precursors of T cell populations as opposed to total proliferative responses. Limiting dilution analyses are conducted at approximately one responding cell per culture well. Pure populations of antigen-specific T cells re-stimulated in vitro by parasite antigens (in excess TCGF) can be used to assess the cellular immunogenicity of parasite antigens. These procedures will determine the relative immunogenicity of parasite antigens independent of the in vitro culture phenomenon. The three main categories of T cell functions are cytotoxic/killer, helper, and suppressor activities. The involvement of major histocompatability complexes (MHC) in parasitic infections is unresolved although it appears T cell killers function only against targets carrying MHC differences. Assessment of potential T cell killer/cytotoxicity activity in parasitic infections is difficult. A suggested alternative is measurement of total cytotoxic T cells in a population b y techniques such as modified chromium release assays. The level of T cell helper activity attributed to parasite antigenactivated T cells can be assessed by systems utilizing culture of parasite antigens, antigen-specific T cells, B cells, and unrelated antigens such as sheep red blood cells (SRBC) or 2,4,6-trinitrophenyl (TNP)--SRBC. Assessment of suppressor cell activity (T cells or T cell products) involves measuring suppressed responses to unrelated antigens such as SRBC. Immunoglobulin E (IgE) production is a characteristic part of host reactions to parasites. Recently it has been shown that in the parasitized host, there are T and B lymphocytes that synthesize lymphokines that can stimulate (and potentiate) IgE production. In this issue, Urban addresses the cellular basis of IgE responses as it relates to surface IgE-bearing B lymphocytes (SIgE-cells), precursors of IgE-containing plasma cells, and the influence of T cells on the development of IgE-containing cells after infection. The conversion of SIgM cells to SIgE--SIgM double bearing cells by a factor from B cells suggests that SIgE--B cell expression is not T cell dependent. Precursors of the IgE-containing cells are predominantly SIgE--SIgM double-bearing cells. IgE B-memory cells are mostly SIgE--SIgM

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114 double-bearing cells and most B-memory cell precursors of IgM or IgGcontaining cells do not bear SIgE. The majority of SIgE cells are likely to be SIgE-, SIgM-, and SIgD-triple bearing cells that develop into IgEcontaining cells through T-dependent processes. T cells are the source of IgE-potentiating factor and they provide antigenspecific helper function for development of B-memory cells to IgM-, IgG-~ and IgE-containing cells. T cell replacing factor (TRF) provides helper T cell activity. Following Nippostrongylus braziliensis-infections, sub-populations of T cells express Fc receptors that spontaneously release an IgE-potentiating factor which binds to SIgE and enhances development of antigen stimulated SIgE-cells to IgE-containing cells. Immunodeficiency models have been used in the characterization of immune responses to parasites. An overview is presented (Jacobson) of the selective use of immunosuppressive agents and genetically immunodeficient animals. Although chemical immunosuppressants and irradiation techniques are often used, their effects are generally indiscriminate. With the use of steroids, the separation of anti-inflammatory effects from immunosuppressive effects is difficult. Alkylating agents such as cyclophosphamide (Cy) also have limited usefulness due to selective toxicity for B cells and inhibition of T cell dependent activities. Irradiation can provide incompetent hosts as cell recipients in adoptive cell transfer studies although there are many secondary effects of radiation. Congenitally immunodeficient models (nude mice, Biozzi mice and w/w v anemic mice) are quite useful because of the inability of their immune systems to react in certain ways. Selective immunodepression induced by specific antisera provides an extremely useful technique for evaluating effects of parasites on host immunity; the advent of monoclonal antibodies will expand the potential of this type of immunosuppression. Because of the non-specific activity of antilymphocyte serum (ALS), results from its use must be interpreted with caution. Host-specific anti-eosinophil serum (AES) is expected to be quite useful. Antisera against Ig-heavy chains induce in vivo as well as in vitro suppression (isotype immunosuppression). Although experiments using anti-isotype serum are involved and demanding, the resulting B cell deficiencies can be quite useful in explaining B cell function. Recently acquired knowledge about the pathways involved in the development of inflammation, particularly the complement cascade and coagulation, has stimulated parasitologists to re-evaluate previous explanations of the host--parasite interface. Although the reports of the interaction between inflammatory pathways and helminths are limited as reviewed here (Leid), they are sufficient to warrant further experimentation. The ability of parasites to inhibit the activated Hageman Factor of the intrinsic coagulation pathway, an early blockage, could explain the lack of coagulation otherwise expected with pathological insults as severe as those induced by parasites. Complement plays an important role in the resistance to some parasites and now there is evidence suggesting that certain parasites are able to mod-

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ulate the complement cascade. The ability to inactivate or inhibit such functional cell products further demonstrates the methods by which parasites may potentially modulate inflammatory processes that would otherwise adversely affect them. These proposed and documented methods of parasitedependent modulation of acute inflammation should promote further interest and research. Immunodiagnosis of parasitic infections requires the use of adequate quantities of parasite antigens that have been purified, identified, and characterized. Tsang reviews new methods of purification and identification of diagnostic antigens. Schistosoma spp. models are used to demonstrate high yields of low cross-reactive urea solubilized antigens. For diagnostic purposes, the elimination of cross-reactive components is considered more important than the isolation of a single molecular species antigen. The previously described single-tube kinetic-dependent enzyme-linked immunosorbent assay (k-ELISA) has application as a qualitative and quantitative assay since in the k-ELISA only one rate-limiting step is present. The exposure of parasite surface antigens to the host immune system dictates the importance of their identification and analysis. Approaches to surface antigen analysis are reviewed (Barbet). Surface proteins and glycoproteins can be radio-labeled by techniques using reagents that do not penetrate parasite surface membranes. Identification and characterization of these labeled molecules are best accomplished by a combination of isoelectric focusing in one direction and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) in the other direction. Monoclonal antibodies, when combined with these analytical techniques, provide the most advanced methods of parasite surface antigen identification. Following initial identification, quantities of surface antigens often need to be harvested and purified. Surface antigens can be removed and solubilized by various combinations of physical, ultrasonic, and detergent disruption. Solubilized antigens can be purified by techniques such as gel filtration, ion exchange chromatography, isoelectric focusing, preparative gel electrophoresis, high pressure liquid chromatography, and affinity chromatography (monoclonal antibodies can be covalently attached to cyanogen bromide activated Sepharose). To detect antigenic variations between parasites or various isolates, identified and purified surface antigens can be analyzed by techniques including various radioimmunoassays, immunofluorescence, peptide mapping and sequencing, and X-ray crystallography. The first exposures of the host immune system to parasite antigens include those from the surfaces of infective third stage larvae (L3). These antigens must be isolated and characterized so that their role in host immune responses can be evaluated. Murrell and Graham report their solubilization studies on L3 epicuticular antigens of Strongyloides ratti. Ferritinconjugated antibody and indirect fluorescent antibody (IFAT) techniques can be used to label these antigens which are then detected with an electron microscope. Even limited extraction and solubilization is difficult using

116 routine techniques with detergents, KC1, organic solvents, freeze-thawing, and enzymes. Although many parasite antigens have been considered fragile, it is evident that at least some of the somatic (epicuticular) antigens are quite resistant, stable, and tightly bound. The lack of species specificity and the somewhat limited protective quality of the epicuticular antigens raise questions as to the relation of these antigens to protective immunity. Many sophisticated analytical techniques have been developed in recent years. Adaptation of these procedures for immunoparasitology research has been complicated by many technical problems related to the unique structures, life cycles, and metabolism of parasites. Hayunga and Murrell have reviewed some of these problems encountered in the radiolabeling of surface antigens on helminth parasites. Two important steps in characterization of helminth surface antigens are labeling of the surface molecules and subsequent extraction of the labeled components. Some culture antigens (exc r e t o r y - s e c r e t o r y products-ESP, exoantigens, or metabolic antigens) may contain surface antigens shed during membrane turnover. In using harsh techniques for extracting surface antigens, contamination by various internal proteins or ESP must be considered. Clearly, subtle things such as improper incubation (in buffers such as phosphate-buffered saline as opposed to balanced salt solutions) can cause artifacts by leakage of internal proteins. Temperature changes may even cause vomiting of worm gut contents. Lactoperoxidase-catalyzed iodination (12sI) is one m e t h o d of choice for direct surface protein labeling and other methods include the use of diazotized 12sI-iodosulfanilic acid, chloramine-T, and 12sI-iodinated P-hydroxy. phenylpropionic acid, N-hydroxysuccinimide ester. Because of differences between species as well as stage-specific surface antigens, it is obviously important to know the amino acid (AA) content (and sequence?) of the specific surface antigens, particularly since the various labeling techniques involve reactions with specific AA. Still other useful approaches involve labeling of precursors and lectin binding. The Schistosoma mansoni model provides insight into the various problems likely to be encountered in similar studies of other species. Excretory--secretory products (ESP), also called secretory antigens, are the most immunologically active antigens of parasite origin and have been shown capable of stimulating as well as suppressing host immune responses. Many standard biochemical techniques are employed in the characterization of these antigens. Among the most useful analytical separation techniques is high performance (often called pressure) liquid chromatography (HPLC). Use of HPLC in parasitological research has been limited because of technical problems including bed volume overloading (including clogging due to the complex nature of antigens) and improper molecular weight ranges for parasite antigens. Commercial columns are now available which allow HPLC fractionation of even complex parasite antigens without destroying their functional antigenic character. Sogandares-Bernal et al. and Moore et al. have described herein the use of HPLC for characterization of antigens of

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Mesocestoides corti tetrathyridia and Giardia lamblia trophozoites. It is suggested that HPLC may become one of the best preparative fractionation procedures although SDS-PAGE provides more resolving power. Increased sensitivity (detection of lower antigen concentrations) of HPLC is possible if fluorescent labeling of components can be achieved. These reports support the use of combined analytical techniques. As indicated in previous reports, the combination of isoelectric focusing in one direction, and SDS-PAGE in the other, may provide the most resolving power currently available for separation of complex parasite antigens. As agents that influence immunoregulatory mechanisms, immunopotentiators and adjuvants have been useful in inducing viral, bacterial, and t u m o r immunity. Such uses and the limited reports of such agents enhancing the immune responses against parasites have p r o m o t e d further investigations. Klesius reviews the current knowledge of immunopotentiation against internal parasites. Examples include dialyzable leucocyte extracts DLE) containing transfer factor TF) which promotes immunity to Eimeria spp., Trichostrongylus axei, Haemonchus contortus, and Ostertagia ostertagi; TF is thought to function in T cell activation. There is evidence of nonspecific potentiation of immunity against Plasmodium spp., Babesia spp., and Schistosorna mansoni by Bacillus Calmette-Guerin (BCG). Corynebacterium parvum is also suggested as a potentiator of immunity against Trypanosoma cruzi, Toxoplasma gondii, Babesia spp., and Plasmodium spp. Further evidence suggests that various bacterial vaccines (BCG, C. parvum, Brucella abortus S-19, and Coxiella burnetti) are effective as immunopotentiators rather than protective antigens with cross-reactivity. Substances such as muramyl dipeptide (MDP) are thought to function by stimulation of mononuclear phagocytic cells. Levamisole acts as an i m m u n o m o d u l a t o r by stimulation of cell-mediated immune responses. The mechanisms of action of adjuvants in vaccinations against parasitic disease are thought to be the same as in bacterial and viral vaccinations. Evidence of success of immunopotentiation as summarized in the review should serve to promote and direct further research in this area. Development of vaccines against parasites is obviously a complex task requiring a more complete understanding of host--parasite immune interactions In these proceedings, Knopf first discusses the philosophy of h o w non-permissive hosts can be used to evaluate such interactions and then exemplifies such with the Rattus norvegicus--Schistosoma rnansoni model system. These approaches have shown that T cells, antibody, and thyroid hormones are active in the protective immune responses against S. mansoni, and that they are effective at different developmental stages of the fluke. Immunological control of such multistage parasites will most likely require a combination of strategical approaches. The applications of recombinant DNA-monoclonal antibodies-genetic engineering technologies are becoming the most useful approaches to immunology. Various monoclonal antibody techniques are being used to evaluate and describe the mechanisms of host immune responses. The success

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of protective immunity induced by "viral antigens" produced by these techniques is spreading to immunoparasitology research. The involved procedures have been established and references are provided in the text by Knopf. To describe this antigen production, in oversimplified terms, the DNA coded for antigen (protective) amino acid sequences is inserted into an appropriate plasmid promotor site for mRNA transcription. Recombinant plasmids are incorporated into bacteria that, when cultured, will produce the resulting protective antigens that can be used as vaccines.

GARY L. ZIMMERMAN Chairman, Immunoparasitology Symposium

(School of Veterinary Medicine, Oregon State University, Corvallis, Oregon 97331-4802, U.S.A.)